We analyzed and compared the difference in sinapine concentration in rapeseed meal between the filamentous fungus, Trametes sp 48424, and the yeast, Saccharomyces cerevisiae, in both liquid and solid-state fermentation. During liquid and solid-state fermentation by Trametes sp 48424, the sinapine concentration decreased significantly. In contrast, the liquid and solid-state fermentation process by Saccharomyces cerevisiae just slightly decreased the sinapine concentration (P ≤ 0.05). After the solid-state fermented samples were dried, the concentration of sinapine in rapeseed meal decreased significantly in Saccharomyces cerevisiae. Based on the measurement of laccase activity, we observed that laccase induced the decrease in the concentration of sinapine during fermentation with Trametes sp 48424. In order to eliminate the influence of microorganisms and the metabolites produced during fermentation, high moisture rapeseed meal and the original rapeseed meal were dried at 90 °C and 105 °C, respectively. During drying, the concentration of sinapine in high moisture rapeseed meal decreased rapidly and we obtained a high correlation coefficient between the concentration of sinapine and loss of moisture. Our results suggest that drying and enzymes, especially laccase that is produced during the solid-state fermentation process, may be the main factors that affect the concentration of sinapine in rapeseed meal.

In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid acyltransferases from N. benthamiana.

Rapeseed meal and waste molasses are two important agro-industrial by-products which are produced in large quantities. In this study, solid state fermentation and fungal autolysis were performed to produce rapeseed meal hydrolysate (RMH) using fungal strains of Aspergillus oryzae, Penicillium oxalicum and Neurospora crassa. The hydrolysate was used as fermentation feedstock for heterotrophic growth of microalga Crypthecodinium cohnii that produce docosahexaenoic acid (DHA). The addition of waste molasses as a supplementary carbon source greatly increased the biomass and DHA yield. In the batch fermentations using media composed of diluted RMH (7%) and 1-9% waste molasses, the highest biomass concentration and DHA yield reached 3.43 g/L and 8.72 mg/L, respectively. The algal biomass produced from RMH and molasses medium also had a high percentage of DHA (22-34%) in total fatty acids similar to that of commercial algal biomass. RMH was shown to be rich in nitrogen supply comparable to the commercial nitrogen feedstock like yeast extract. Using RMH as sole nitrogen source, waste molasses excelled other carbon sources and produced the highest concentration of biomass. This study suggests that DHA production of the marine dinoflagellate C. cohnii could be greatly improved by concomitantly using the cheap by-products rapeseed meal hydrolysate and molasses as alternative feedstock.

从可食用的纳豆中筛选出一株能够高效降解黄曲霉毒素B1(aflatoxin B1,简称AFB1)的细菌,该细菌的发酵上清液经浓缩后制成的粗酶液对AFB1降解率达到91.4％.对该菌进行了分类地位鉴定并初步研究了粗酶液的酶学性质.结果表明,该菌经生理生化和16S rDNA序列比对鉴定为枯草芽孢杆菌,并命名为Natto3.Natto3的粗酶液降解AFB1的最适培养时间为72h,最适反应温度为37℃,最适pH值是8.5,Zn2+、Mn2+、Mg2+、Cu2+、Li+五种金属离子均会不同程度地抑制降解活性.此外,将该粗酶液添加在被黄曲霉毒素高度污染的花生样品中进行脱毒实验,可使花生中AFB1的浓度从192μg/kg降至43μg/kg.

Long-chain acyl-CoA synthetase (ACSL; EC 6.2.1.3) catalyzes the conversion of free fatty acid to acyl-CoA ester, which is necessary for many pathways of fatty acid and lipid metabolism. The diatom Phaeodactylum tricornutum genome encodes five putative ACSLs (PtACSL1-5) that contain several highly conserved motifs and share limited sequence similarities with each other and with other known ACSLs. To verify their long-chain acyl-CoA synthetase activities, five cDNAs encoding these PtACSLs were cloned, expressed, and tested for their ability to complement the Saccharomyces cerevisiae double mutant FAA1ΔFAA4Δ. Only two of five PtACSLs were able to restore growth, facilitate exogenous fatty acid uptake, and enhance storage lipid accumulation. We also found that P. tricornutum cells are capable of importing long-chain fatty acids from extracellular environment. The identification of P. tricornutum ACSLs will provide molecular basis for the study of ACSL-mediated lipid synthesis and metabolism in diatoms.

Sinapine is an important secondary metabolites in cruciferous plants. Rapeseed is the widest planted crops containing sinapine. Sinapine is the most important simple phenolic compounds in rapeseed meal which affects the palatability, protein digestion and absorption of rapeseed meal and the odor of eggs. The existing form, synthesis pathway, property,extraction and purification methods and position in phenolic compounds of rapeseed and rapeseed meal of sinapine, its impact on nutritional value of rape-seed meal and its degradation under physicochemical and enzymatic actions were reviewed, so as to pro-vide a suitable method for reducing sinapine content in rapeseed meal.

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have received growing attention due to their significant roles in human health. Currently the main source of these nutritionally and medically important fatty acids is marine fish, which has not met ever-increasing global demand. Microorganisms are an important alternative source also being explored. Although many microorganisms accumulate omega-3 LC-PUFAs naturally, metabolic engineering might still be necessary for significantly improving their yields. Here, we review recent research involving the engineering of microorganisms for production of omega-3 LC-PUFAs, including eicospentaenoic acid and docosohexaenoic acid. Both reconstitution of omega-3 LC-PUFA biosynthetic pathways and modification of existing pathways in microorganisms have demonstrated the potential to produce high levels of omega-3 LC-PUFAs. However, the yields of omega-3 LC-PUFAs in host systems have been substantially limited by potential metabolic bottlenecks, which might be caused partly by inefficient flux of fatty acid intermediates between the acyl-CoA and different lipid class pools. Although fatty acid flux in both native and heterologous microbial hosts might be controlled by several acyltransferases, evidence has suggested that genetic manipulation of one acyltransferase alone could significantly increase the accumulation of LC-PUFAs. The number of oleaginous microorganisms that can be genetically transformed is increasing, which will advance engineering efforts to maximize LC-PUFA yields in microbial strains.

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host.

The sulfonated azo dye Reactive Orange 16 is the commonly used representative of reactive dyes, but is hard to be degraded by some conventional treatment methods. In order to develop more efficient and more cost-effective treatment methods for degrading this recalcitrant dye, the capability of the white-rot fungus Ganoderma sp. En3 isolated by our laboratory to decolorize and detoxify Reactive Orange 16 was investigated in this study. Ganoderma sp. En3 had a strong ability to decolorize high concentrations of Reactive Orange 16 and simulated textile wastewater containing Reactive Orange 16 in submerged cultures. Decolorization of Reactive Orange 16 and its simulated dye effluents by this fungus resulted in the decrease of phytotoxicity. Ganoderma sp. En3 had strong adaptability and tolerance to high concentrations of Reactive Orange 16. Compared with some previous research, Ganoderma sp. En3 was superior to some other fungal strains reported previously in the rate and extent of decolorizing Reactive Orange 16. It was also found that the real textile wastewater could be efficiently decolorized by Ganoderma sp. En3 in submerged cultures. The crude enzyme produced by Ganoderma sp. En3 could also efficiently decolorize Reactive Orange 16 and simulated textile wastewater under in vitro conditions. (C) 2013 Elsevier B.V. All rights reserved.

The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off-line 2D system coupling of nonaqueous RP and silver-ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.

We are developing a collection of Bacillus strains, isolated from different environments, for use in controlling Sclerotinia sclerotiorum on oilseed rape in China and elsewhere. Strain BY-2, isolated from internal tissues of an oilseed rape root, was demonstrated to be Bacillus subtilis based on biochemical and morphological characteristics and on 16S RNA gene sequence. Photographic evidence from gnotobiotic studies using the lacZ-tagged strain BY-2(pUC18) confirmed that this strain was capable of colonizing internal root tissues. Strain BY-2 did not effectively colonize the ectorhizosphere or the surface of the stems or leaves of oilseed rape when applied in pellet or wrap seed treatment formulations. Populations of BY-2 dropped from 10(8) CFU seed (1) to 104 CFU g root (1) and <= 10(2) CFU g stem (1) or leaf (1) after 60 days. Strain BY-2 was applied as a pellet seed treatment formulation alone, as a spray at flowering alone, and as the pellet seed treatment formulation combined with the spray application in two field trials at the Wuxue location conducted in two consecutive years. These three treatments containing BY-2 provided disease control (disease incidence) and mean seed yield that was similar to the chemical control treatment and significantly greater than the pellet without bacteria and non-treated control treatments. All three of these BY-2 treatments were similar to each other with regard to these two metrics and to treatments containing B. subtilis Tu-100, a genetically distinct strain previously shown to be effective against this disease. In two additional field trials, conducted in consecutive years at the Wuhan location, strain BY-2 applied as a wrap seed treatment formulation alone, as a spray at flowering alone, and as the wrap seed treatment combined with the spray application provided disease control (disease incidence) and mean seed yield that was similar to the chemical control treatment. These three BY-2 treatments also were significantly greater than the non-treated control treatments and compared favorably to treatments containing Tu-100. There was no evidence of BY-2 promoting growth of oilseed rape when applied in the pellet or wrap seed treatment formulations in field trials conducted at the Wuxue or at the Wuhan locations. We now have three Bacillus strains (B. subtilis strains BY-2 and Tu-100, B. megaterium A6) that control S. sclerotiorum on oilseed rape in the field that can be tested in strain combinations for enhanced disease control. We also have multiple methods for application of Bacillus strains as both seed treatment and foliar applications were effective. Published by Elsevier Inc.

In oleaginous fungus Cunninghamella echinulata, Δ9-fatty acid desaturase introduces the first double bond into a saturated fatty acid. Three distinct genes, designated as d9dma, d9dmb and d9dmc, all encoding putative Δ9-fatty acid desaturases were isolated from this strain. The predicted proteins showed 79–87 % identity to other fungal Δ9-fatty acid desaturases. They all contain three conserved histidine boxes, C-terminal cytochrome b 5 fusion and four transmembrane domains characteristic of Δ9-desaturase. Each putative Δ9-desaturase gene from C. echinulata was able to complement the ole1 mutation in Saccharomyces cerevisiae L8-14C through heterologous expression. Analysis of the fatty acid composition of the transgenic yeast revealed that the conversion rates of 16:0 and 18:0 by D9DMA were obviously higher than those of D9DMB and D9DMC. In addition, D9DMA, D9DMB and D9DMC all had a substrate preference for 18:0 compared with 16:0. Of interest, D9DMA could saturate 12:0, 14:0, 16:0, 17:0, 18:0 and 20:0, while D9DMB saturated 14:0, 16:0, 17:0, 18:0 and 20:0. We also noticed that the transcriptional level of d9dma in C. echinulata was stimulated by cell growth but not by decline in temperature. In contrast, expression of d9dmb and d9dmc was regulated by neither cell growth nor decline in temperature in this strain.

Culture conditions were adapted to obtain larger biomass and higher docosahexaenoic acid(DHA)yield of Crypthecodinium cohnii under flask fermentation level.Results showed that the optimal culture should be at 22℃,in carbon-source culture with 12g/L glucose for 4d and then shifted to the same media but with 3g/L sodium acetate instead for 4d.Under this optimal condition,the C.cohnii biomass yield,DHA yield and DHA content reached 234.85mg/(L·d),17.45mg/(L·d),74.36mg/g dry weight respectively.

In modern diet of human being, the ω-6/ω-3 ratio of dietary fatty acid was shifted much towards ω-6 series. In this work, a fatty acid desaturase (FAD) gene lkfad15 was cloned and identified from oleaginous yeast Lipomyces kononenkoae . Function analysis results shown that LKFAD15 is a novel Δ12/Δ15 bifunctional FAD which could not only produce linoleic acid and α-linolenic acid use just oleic acid as substrate but also adjust the ω- 6/ω-3 fatty acids ratio to the WHO recommended ratio. Phylogenetic analysis of LKFAD15 suggested it is a specific intermediate product of gene evolution derives from independent gene duplication events.

The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) that are important to human health. Here, we report a functional characterization of a Δ4-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops.

Diacylglycerol acyltransferase (DGAT) plays a pivotal role in triacylglycerol (TAG) formation in some oleaginous organisms. We describe here the identification of a type 2 DGAT (PtDGAT2B) in the diatom Phaeodactylum tricornutum that contains four putative type 2 acyl‐CoA:DGATs, sharing little sequence similarity with each other. TAG synthesis and lipid body formation could be completely restored in a Saccharomyces cerevisiae TAG‐deficient quadruple mutant by expressing PtDGAT2B. Up‐regulation of PtDGAT2B precedes the accumulation of TAG. Functional analysis of enzyme activity in vivo demonstrated that expression of PtDGAT2B can increase the proportion of unsaturated C16 and C18 fatty acids in yeast TAG.

Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu2+ and Fe2+ could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.

Laccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus in Pichia pastoris can significantly enhance the resistance of yeast to H(2)O(2)-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H(2)O(2) and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression in Pichia pastoris could increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene in Pichia pastoris was found to be enhanced by the oxidative stress caused by exogenous H(2)O(2). The stimulation of laccase gene expression in response to exogenous H(2)O(2) stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, including PpYAP1, PpGPX1, PpPMP20, PpGLR1, and PpGSH1. Taken together, these results suggest that the expression of the laccase gene in Pichia pastoris can enhance the resistance of yeast to H(2)O(2)-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage.

Microwave-assisted enzymatic hydrolysis conditions for cottonseed meal were optimized.Through single factor experiments,the optimal microwave-assisted alkali-protease hydrolysis conditions were determined as follows:microwave temperature 60 ℃,microwave power 500 W,enzyme dosage 5%(based on the mass of substrate),time 15 min.The optimal microwave-assisted flavourzyme hydrolysis conditions were as follows:microwave temperature 60 ℃,microwave power 600 W,enzyme dosage 5%(based on the mass of substrate),time 15 min.According to the above conditions,the cottonseed meal was continuous hydrolyzed with microwave-assisted,and the peptide content of hydrolysates was 13.32 mg/mL.Functional properties of hydrolysates were greatly improved,such as oil absorption,foaming, emulsifying.

Upstream sequence and partial coding sequence(CDS) of the phosphoglycerate kinase(PGK) gene were cloned from oleaginous yeast Trichosporon sp.by degenerate PCR and chromosome walking.The computation analysis revealed that a promoter-like sequence in the upstream region of the PGK coding region including essential elements such as TATA BOX and CAAT BOX was cloned.The promoter-proximal region cloned was fused with the coding region of the hygromycin gene,and then cloned into the expression vector pTF-PHT.The recombinant vector was transformed into Trichosporon fermentans,and the transformed yeast was able to grow on the selective agar containing hygromycin.The results indicated that the promoter-proximal region of the phosphoglycerate kinase gene of Trichosporon sp was able to drive the expression of heterologous genes in the Trichosporon fermentans,which provides new avenues for developing novel engineering yeast or yeast expression system.